SHG44人脑胶质瘤干细胞的提取、纯化与鉴定

Extraction,purification and identification of SHG44 human brain glioma stem cells

目的:探讨SHG44人脑胶质瘤干细胞的提取、纯化与鉴定方法。方法:采用以6孔悬浮细胞培养板为载体的无血清DMEM/F12培养基(含EGF、FGF、B27)提取与纯化SHG44人脑胶质瘤干细胞,通过CD133联合Nestin标记进行免疫荧光鉴定,并在倒置显微镜下观察细胞形态及干细胞球的形态、大小及数目。结果:SHG44人脑胶质瘤干细胞能在含无血清DMEM/F12培养基的6孔悬浮细胞培养板中存活、增殖并形成干细胞球;悬浮培养3d初步形成呈巢状悬浮生长的胶质瘤干细胞团,7d后干细胞球大小、形态基本不变。经统计分析,悬浮培养板培养纯化的胶质瘤干细胞球,随着纯化次数增加,胶质瘤干细胞球更大、数目更多(P<0.05),且呈更规则球形。结论:本研究成功提取、纯化、鉴定出SHG44胶质瘤干细胞,并进行传代扩增培养;Cyagen悬浮细胞培养板可作为筛选胶质瘤干细胞的理想载体。

Objective:To extract,purify and identify the SHG44 human brain glioma stem cells.Methods:SHG44human brain glioma stem cells were cultured in serum-free DMEM/F12 medium supplemented with EGF,FGF and B27.The 6-well suspension cell culture plates were used.The cells were identified by examining the expressions of CD133 and Nestin with immunofluorescence staining method,and the morphology,size and number of the stem cell balls were observed under the inverted microscope.Results:The SHG44 human glioma stem cells survived,proliferated and formed stem cell balls in the culture plates.The cells formed nest-like balls of glioma stem cells in suspension after 3days,and unchanged for the size and morphology after 7days.It was statistically found that with the increase of number of purification,more glioma stem cell balls were formed with larger and more regular shape.Conclusion:The SHG44 glioma stem cells were successfully extracted,purified and identified,and expanded with passages.Cyagen suspension cell culture plate is ideal for screening glioma stem cells.